Non-denaturing purification of alpha-Synuclein from erythrocytes

Abstract

Pathogenic aggregation of α-Synuclein (αSyn) is implicated in familial and sporadic Parkinson disease (PD) and several other synucleinopathies. Choosing non-denaturing conditions, we developed a several purification schemes in order to obtain αSyn from human erythrocytes, enabling researchers to employ biophysical techniques in order to elucidate its native structure and properties.

Introduction

Here we introduce a novel way of purifying human α-Synuclein from human erythrocytes, leaving the physiological conformation of the protein intact. This methology has been also successfully applied to human neuroblastoma cells stably overexpressing wt human α-Synuclein as well as to COS-7 cells transiently overexpressing wt and mutant forms of α-Synuclein. The overall purification is divided in three general steps: An ammonium sulfate preciptation giving a crude protein solution, a bulk purification which can be adjusted to the exact sample properties (volume, cell type, cross contamination) utilizing alternatively 3 different methods (AX, HIC, covalent chromatography), and a polishing step giving pure protein after size exclusion chromatography (Fig. 1).

Only for erythrocyte lysate: Wash pellet 5-10x with 55% (NH4)2SO4 in dd water, (calculation via http://www.encorbio.com/protocols/AM-SO4.htm), until red color of sup is mostly gone. Washing means nutating the pellet in 55% (NH4)2SO4 for 10 min at 4 °C, then spinning it down at at 20,000 g for 20 min, keeping the pellet and discarding the supernatant.

Bring washed pellet up in respective chromatography binding buffer (see below) by nutating it for 1 h at 4 °C

Troubleshooting

A. Instead of freshly drawn blood, older samples can be employed. The ratio of α-Synuclein/Hemoglobin in those samples can be drastically reduced however, making the purification more challenging

3D5 cells:

A. It has to be made sure that the cell lysate does not heat up too much during sonication. Short sonication steps with incubation of the sample on ice in between are therefore recommended.

B. In cell culture cell lysates, a 25% (NH4)2SO4 pre-precipitation step can be beneficial to remove unwanted protein. If HIC is employed for the bulk purification, this is not necessary since the respective binding buffer already contains approx. 25% (NH4)2SO4 and thereby eliminates contamination before the column is loaded

Ammonium sulfate precipitation:

In erythrocyte lysate, washing the 50% (NH4)2SO4 pellet in 55% (NH4)2SO4 solution is highly recommended. Since Hemoglobin stays soluble until 60-65% (NH4)2SO4, the washing steps provide a cheap and easy method to eliminate large amounts of contaminating Hemoglobin.

Bulk purification

The exact method of bulk purification has to be decided by the nature of the sample. In general, the HIC gave the highest purity but lowest yield. If high trough-put is needed, the Sepharose 6B method should be applied. If large amounts of Hemoglobin are present, AX is recommended. If a particular sample has low purity with one of the chosen methods, an additional step utilizing one of the other methods can be employed.

A: HIC advantages: Fast, cheap, best purity, (NH4)2SO4 in binding/elution buffer stabilizes folding. Disadvantages: Low binding capacity, struggles with high amounts of contaminating Hemoglobin. Results can be inconsistent depending on the exact sample. Has sometimes to be applied twice to effectively purifiy all α-Synuclein out of solution.

B: AX advantages: Fast, cheap, good binding affinity, Hemoglobin does not bind (making it very effective in separating α-Synuclein and Hemoglobin). Disadvantages: High amounts of NaCl in elution buffer can destabilize α-Synuclein, causing it to precipitate. Transthyretin and α-Synuclein elute very close to each other making thorough removal of blood plasma prior to erythrocyte lysing necessary. Has sometimes to be applied twice to effectively purifiy all α-Synuclein out of solution.

C: Instead of Sepharose 6B gel medium, Sepharose 4B can be used. Sepharose 6B has shown to have a better binding capacity, while Sepharose 4B packed columns can be easier reactivated.
Sepharose 6B advantages: Simple and consistent, good for large sample volumes, high yield, removes both Transthyretin and Hemoglobin from erythrocyte lysate samples very effectively. Since no high pressures/high flow rates, no buffer gradient and no monitoring of UV-absorption or conductivity are needed, it can be done with a simple peristaltic pump. Disadvantages: Expensive gel media, no pre-packed columns commercially available, regeneration of column time consuming.

Size Exclusion Chromatography (Superdex 200):

Instead of a Superdex 200 column, Superdex 75, Superose 12 and Sephacryl 200-HR were also successfully employed. The advantage of the Superdex 200 gel medium is the ability to separate Hemoglobin from α-Synuclein in contrast to the above mentioned media.

Anticipated Results

Yield

From 40 ml of freshly drawn packed red blood cells, up to 0.5 mg α-Synuclein can be recovered.

2SO4 precipitation

As seen in Fig. 2, α-Synuclein usually precipitates between 30 and 45% (NH4)2SO4. Human Hemoglobin should stay soluble until 60%.

Covalent chromatography (Sepharose 6B, XK 16/100): α-Synuclein should be completely found in the clear flow-trough. Hemoglobin should be completely bound to the column and then be eluted with DTT (Fig. 4).

C: Photography of different aliquots of Sepharose 6B purification scheme visualizing the stepwise removal of Hemoglobin from the sample.

Purity

Example Coomassie stains of the different purification steps for human erythrocytes are shown in Fig. 5. After applying the procedure the purity of the sample should be >95% (measured by densitometry of Coomassie stain).

Figure 5: Coomassie stain of different purification steps

SDS-PAGE Coomassie stain analysis of aliquots taken from different stages of purification.